DIFFERENTIAL STAT5 ACTIVATION AND PHENOTYPIC MARKER EXPRESSION BY IMMUNE CELLS FOLLOWING LOW LEVELS OF ETHANOL CONSUMPTION IN MICE

ABSTRACT

Ethanol has been recognized as an immunosuppressive agent for many years. Effects of high levels of ethanol consumption on immune functions have been extensively studied, but little is known about the effects of low levels (scuh as 5% ethanol) of ethanol consumption. Herein we report that exposure of mice to 5% ethanol for 4–8 weeks decreases IL-2-augmented splenic NK cell activity, decreases the numbers of NK cells in spleen and liver, decreases the number of granulocytes (Gr-1⁺) in bone marrow and spleen, and decreases the percentages of B cells in liver. In contrast, the percentages of CD4⁺CD8⁺ thymocytes, CD4⁺CD8^? splenocytes, CD4⁺CD8^? liver nonparenchymal cells, CD3⁺ splenocytes, and CD3⁺ bone marrow cells were increased. Furthermore, exposure to 5% ethanol increases STAT5 activation in T cells and liver cells while decreases STAT5 activation in NK cells. Taken together, these findings suggest that low levels of ethanol consumption can differentially modulate immune cells in thymus, spleen, bone marrow and liver, which may be due to differential regulation of STAT5 activation by ethanol.     

B Cells Modulate Systemic Responses to Pneumocystis murina Lung Infection and Protect On-Demand Hematopoiesis via T Cell-Independent Innate Mechanisms when Type I Interferon Signaling Is Absent

HIV infection results in a complex immunodeficiency due to loss of CD4⁺ T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag^−/−) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR^−/−) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag^−/− mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag^−/− mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4⁺ T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.     

Differential STAT5 activation and phenotypic marker expression by immune cells following low levels of ethanol consumption in mice

Ethanol has been recognized as an immunosuppressive agent for many years. Effects of high levels of ethanol consumption on immune functions have been extensively studied, but little is known about the effects of low levels (scuh as 5% ethanol) of ethanol consumption. Herein we report that exposure of mice to 5% ethanol for 4-8 weeks decreases IL-2-augmented splenic NK cell activity, decreases the numbers of NK cells in spleen and liver, decreases the number of granulocytes (Gr-1⁺) in bone marrow and spleen, and decreases the percentages of B cells in liver. In contrast, the percentages of CD4⁺CD8⁺ thymocytes, CD4⁺CD8^- splenocytes, CD4⁺CD8^- liver nonparenchymal cells, CD3⁺ splenocytes, and CD3⁺ bone marrow cells were increased. Furthermore, exposure to 5% ethanol increases STAT5 activation in T cells and liver cells while decreases STAT5 activation in NK cells. Taken together, these findings suggest that low levels of ethanol consumption can differentially modulate immune cells in thymus, spleen, bone marrow and liver, which may be due to differential regulation of STAT5 activation by ethanol.     

Liver X receptor regulates mouse GM-CSF-derived dendritic cell differentiation in vitro

Liver X receptors (LXRs) are nuclear receptors that play an essential role in lipid and cholesterol metabolism. Emerging studies indicate a potential function for LXRs in regulating dendritic cell (DC)-dependent immune responses; however, the role of LXRs in DC differentiation is largely unknown. Here, we report that LXRα regulates the differentiation of mouse GM-CSF-derived DCs. Activation or overexpression of LXRα significantly enhanced myeloid DC differentiation from mouse bone marrow (BM) cells, while siRNA-mediated knockdown of LXRα suppressed DC differentiation. In addition, we demonstrated that LXR agonist-programmed DCs showed an increased capacity for stimulating T-cell proliferation. Mechanistic studies showed that activation of LXR could inhibit the phosphorylation of STAT3 and downregulate the expression of its target, S100A9, an important negative regulator of myeloid DC differentiation. We also found that Histone deacetylase (HDAC) inhibition interfered with the effect of LXR on STAT3 signaling via acetylation of STAT3. Chromatin immunoprecipitation assays confirmed that LXR activation and HDAC inhibition balanced the recruitment of STAT3 to the S100A9 promoter, which involved distinct post-translational modifications of STAT3. In conclusion, our observations demonstrated a novel role for LXRα in GM-CSF-derived DC differentiation and revealed the underlying mechanism.     

Regulation of dendritic cell differentiation and function by Notch and Wnt pathways

Summary: The process of dendritic cell differentiation is governed by a tightly controlled signaling network regulated by cytokines and direct interaction between progenitor cells and bone marrow stroma. Notch signaling represents one of the major pathways activated during direct interaction between hematopoietic progenitor cells and bone marrow stroma. Wnt pathway is activated by soluble proteins produced by bone marrow stroma. Until recently, the role of Notch and Wnt signaling in the development of myeloid cells and dendritic cells in particular remained unclear. In this review, we discuss recent exciting findings that shed light on the critical role of Notch and Wnt pathways, their interaction in differentiation and function of dendritic cells, and their impact on immune responses.     

The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4

In the bone marrow, progenitor (pro-) and precursor (pre-) B cells depend on close contact with stromal cells for growth and maturation. Stromal cell-derived factor 1 (SDF-1), also known as pre-B cell growth-stimulating factor, is produced by bone marrow stromal cells and was reported to act together with interleukin-7 as co-mitogen for pre-B cells. SDF-1 was recently shown to be a chemokine which is chemotactic for different types of leukocytes and acts via the chemokine receptor CXCR4. Using sorted B220⁺ bone marrow cells and several B cell lines characteristic for different stages of B lymphopoiesis, we now show that SDF-1 is a potent attractant for pro- and pre-B cells, but is inactive on B cells at later stages of development. In early B cell precursors, SDF-1 induced intracellular Ca²⁺ mobilization and in vitro migration with a potency and efficacy similar to that observed for chemokines acting on blood leukocytes. These responses were mediated via CXCR4 as they could be inhibited by an anti-receptor antibody. SDF-1 is the first chemokine shown to act on early-stage B cell precursors. Mice lacking SDF-1 die perinatally and show a severe deficiency in B lymphopoiesis. We propose that SDF-1 released from the stromal cells exerts its critical hematopoietic function by selectively attracting and confining early B cell precursors within the bone marrow microenvironment that provides the necessary factors for growth and differentiation.     

Genesis of B lymphocytes in the bone marrow: Extravascular and intravascular localization of surface IgM-bearing cells in mouse bone marrow detected by electron-microscope radioautography after in vivo perfusion of 125I anti-IgM antibody

The role of mammalian bone marrow in generating surface IgM (sIgM)-bearing B lymphocytes is reviewed. Precursor cells in the marrow give rise to large, rapidly dividing cells bearing free cytoplasmic μ chains (cμ). The progeny of the large cμ⁺ cells form a population of small, nondividing cμ⁺ cells that mature into small lymphocytes, progressively expressing sIgM and other B-cell surface membrane components. Newly formed sIgM⁺ cells soon migrate through the bloodstream to the spleen and other lymphoid tissues, where they may die after a short lifespan or be activated to produce antibody molecules. The large-scale lymphocytopoiesis in the bone marrow thus maintains a population of rapidly renewed virgin B lymphocytes in the peripheral lymphoid tissues. This process continuously creates and selects B cell clones with the wide range of antibody specificites necessary to mediate primary humoral immune responses through postnatal life. A technique for perfusing radiolabeled anti-IgM antibodies in young mice has now permitted sIgM⁺ cells to be detected radioautographically in histological preparations of bone marrow under the electron microscope. Small sIgM⁺ lymphocytes are situated either singly or in small groups throughout the extravascular hemopoietic compartment of the bone marrow, often near sinusoid walls adjacent to late erythroblasts and reticular cells. Some regional concentrations of sIgM⁺ cells are apparent. sIgM⁺ cells also appear in transit through the sinusoidal endothelium and are markedly concentrated in the lumen of some sinusoids. Intrasinusoidal sIgM⁺ small lymphocytes have high densities of sIgM and long microvilli, on which sIgM molecules are concentrated. These studies reveal the localization and cell associations of specifically identified sIgM⁺ small lymphocytes in the extravascular marrow compartment and suggest that these cells may also undergo a transient intravascular storage and maturation phase. Use of this in vivo immunolabeling technique to detect other cell-surface markers may further elucidate the microenvironmental basis of B lymphocyte genesis in the bone marrow.     

Late stages of hematopoiesis and B cell lymphopoiesis are regulated by α-synuclein,a key player in Parkinson's disease

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein^−/− (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220⁺IgM⁺ B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104 ± 23 × 10⁵ vs. KO: 27 ± 5 × 10⁵). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response.     

MicroRNA-219a-5p suppresses intestinal inflammation through inhibiting Th1/Th17-mediated immune responses in inflammatory bowel disease

MicroRNA (miR)-219a-5p has been implicated in the development of numerous progression of carcinoma and autoimmune diseases. However, whether miR-219a-5p is involved in the pathogenesis of inflammatory bowel disease (IBD) remains elusive. In this study, we demonstrated that miR-219a-5p expression was significantly decreased in the inflamed intestinal mucosa and peripheral blood (PB)-CD4⁺ T cells from patients with IBD. Proinflammatory cytokines (e.g., IL-6, IL-12, IL-23 and TNF-α) inhibited miR-219a-5p expression in CD4⁺ T cells in vitro. Lentivirus-mediated miR-219a-5p downregulation facilitated Th1/Th17 cell differentiation, whereas miR-219a-5p overexpression exerted an opposite effect. Luciferase assays confirmed that ETS variant 5 (ETV5) was a functional target of miR-219a-5p and ETV5 expression was significantly increased in the inflamed intestinal mucosa and PB-CD4⁺ T cells from IBD patients. ETV5 overexpression enhanced Th1/Th17 immune response through upregulating the phosphorylation of STAT3 and STAT4. Importantly, supplementation of miR-219a-5p ameliorated TNBS-induced intestinal mucosal inflammation, characterized by decreased IFN-γ⁺ CD4⁺ T cells and IL-17A⁺ CD4⁺ T cells infiltration in the colonic lamina propria. Our data thus reveal a novel mechanism whereby miR-219a-5p suppresses intestinal inflammation through inhibiting Th1/Th17-mediated immune responses. miR-219a-5p might be a target for the treatment of IBD.     

Reduced TCR‐dependent activation through citrullination of a T‐cell epitope enhances Th17 development by disruption of the STAT3/5 balance

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4⁺ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.     

Notch signaling in differentiation and function of dendritic cells

Hematopoietic stem cells give rise to multiple lineages of cells. This process is governed by a tightly controlled signaling network regulated by cytokines and a direct cell-cell contact. Notch signaling represents one of the major pathways activated during direct interaction between hematopoietic progenitor cells and bone marrow stroma. A critical role of Notch signaling in differentiation of T- and B-lymphocytes has now been established. Until recently, the role of Notch signaling in the development of myeloid cells and particular dendritic cells remained unclear. In this review, we discuss recent exciting findings that shed light on the critical role of Notch in differentiation and the function of dendritic cells and its impact on immune responses.     

CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations

Despite the important role immunoglobulin G (IgG)-secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG-secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG-secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG-secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up-regulation of CD38, and on tonsillar sections, IgG-containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG-secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG-secreting precursor gives rise to a diverse plasma cell compartment.     

Sema4D is required in both the adaptive and innate immune responses of contact hypersensitivity

Originally recognized as a regulator of axon guidance in the nervous system, Semaphorin 4D (Sema4D, CD100) also participates in various immune responses and many immune-related diseases. However, whether Sema4D is involved in the pathogenesis of contact hypersensitivity (CHS) remains unclear. In this study, we explored the role of Sema4D in oxazolone-induced CHS using Sema4D knockout (KO) mice. We found that Sema4D KO mice developed attenuated CHS responses, as indicated by milder ear-swelling, lower expression of IL-1β, IL-6, CXCL2 and CXCL5, and decreased recruitment of neutrophils, CD8⁺ T cells and CD4⁺ T cells. CHS was impaired in the wide type (WT) mice reconstituted with bone marrow from Sema4D KO mice, indicating that deletion of Sema4D gene in hematopoietic cells played a key role in the alleviated CHS in Sema4D KO mice. CHS was also attenuated in the WT mice transferred with draining lymph nodes (dLNs) cells from oxazolone-sensitized Sema4D KO mice, and the activation and differentiation of hapten-specific CD8⁺ T cells were impaired in Sema4D KO mice. Furthermore, Sema4D KO mice expressed less IL-1β and CXCL2 than WT mice after oxazolone sensitization, and after transferred with dLNs cells from oxazolone-sensitized WT mice, naïve Sema4D KO mice showed attenuated CHS responses upon oxazolone challenge, indicating that the innate immune response of CHS in Sema4D KO mice was also abrogated. Taken together, our findings revealed for the first time that Sema4D positively regulated both the adaptive and innate immune responses in CHS.